In vitro cell culture is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment but as close to their natural in vivo conditions as possible. In practice cell culture refers to the culturing of cells derived from multi-cellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi, insects and microbes, including viruses, bacteria and protista.
In vitro cell culture provides material necessary for research and application in pharmacology, physiology, and toxicology. This includes bioprocessing and cell therapy where cell cultures are necessary.
Cells are grown and maintained at an appropriate temperature and gas mixture in a cell incubator. Typically, mammalian cells are incubated at 37° C. with a pH maintained between 7.2 and 7.4. The pH is typically controlled using a bicarbonate buffering system in the medium, in conjunction with an incubator atmosphere of approximately 5-7% carbon dioxide by volume. The carbon dioxide reacts with the water to form carbonic acid which in turn interacts with bicarbonate ions in the medium to form a buffering system which maintains the pH near physiological levels. Oxygen is essential for cellular metabolism and growth. Culture conditions can vary for each cell type, and variation of conditions for a particular cell type can result in different phenotypes. For instance, bicarbonate based buffers can be substituted with mono and di or tri-sodium phosphate buffers, chloride and ammonia buffers, lactate, or organic buffers such as HEPES, etc.
A variety of cell types are grown in vitro and similarly a variety of media are available depending on the particular growth requirements of the cells and the growth conditions.
Monocytes are the largest type of white blood cells (leukocytes) and are an important part of the innate immune system of vertebrates including all mammals. Monocytes are the circulating precursors of macrophages and dendritic cells that migrate from the blood stream across vascular endothelium for immunological surveillance and inflammation response.
Commercially available gas permeable cell culture devices in the form of bags are currently a standard device format used for cell culture. Cell culture bags have the advantage of being disposable, which reduces preparation and clean up time. Additionally, cell culture bags are pre-sterilizable, inexpensive, easy to use and require minimal space for storage and use. Disposables also helps reduce the risk of contamination for the cell culture and for the environment.
Gas permeable cell culture bags are commercially available from OriGen Biomedical Group (OriGen PermaLife™ Bags), Baxter (Lifecell® X-Fold™ related to U.S. Pat. Nos. 4,829,002, 4,937,194, 5,935,847, 6,297,046 B1), Medtronic (Si-Culture™, U.S. Pat. No. 5,686,304), Biovectra (VectraCell™), and American Fluoroseal (VueLife™ Culture Bag System, covered by U.S. Pat. Nos. 4,847,462 and 4,945,203).
For example, one problem of conventional fluoropolymer cell culture bags is that the feed media and cells are mixed within the culture chamber and therefore the amount of media added is limited to the volume of the container. In one example, Matsumiya et al. (U.S. Pat. No. 5,225,346) attempts to correct the problem of media supply by integrating the bag with a medium storage compartment. The culture chamber and medium storage compartment are connected and when fresh medium is needed it is passed from the medium storage compartment to the culture chamber.
Another problem of conventional fluoropolymer cell culture bags is removal of media having diminished nutrient content and increased waste content during cell cultivation which may unavailingly remove cells as well. Additionally, manual manipulation of the culture is generally necessitated to replenish the medium.
Perfusion is a continuous process in which adherent or suspension cell culture is continuously supplied with fresh medium to the bioreactor while spent culture media is continuously harvested. For the latter, cells are continuously filtered from the harvest stream and returned to the bioreactor to maintain a constant culture volume. This continuous process allows the cells to reach high densities as densities of 10-15 million cells/mL can routinely be reached and maintained for extended periods of time. This process is limited to complex perfusion bioreactors that require large volumes of media. In the present embodiments, it is preferred that the cell culture medium above the cell containing compartment is perfused such that the cells are not appreciably moved about.
Therefore, a need exists for a simplified perfusion apparatus that allows removal and replenishment of feed media without diminishing cell count thereby advantageously concentrating cells over a period of time not controlled by feed media supply limitations.